RETRACTED ARTICLE: LncRNA TUG1 was upregulated in osteoporosis and regulates the proliferation and apoptosis of osteoclasts

Blood (5 ml) was extracted from 98 patients with osteoporosis and 60 healthy participants who were admitted to Baoding First Central Hospital from January 2015 to January 2018. Patients with osteoporosis were diagnosed by dual-energy X-ray absorptiometry (T-score of < − 2.5 SD). Blood was used to extract plasma using conventional methods. Inclusion criteria were as follows: (1) patients with osteoclasts who were diagnosed for the first time, (2) patients with complete medical record, and (3) patients who understood the experimental procedure and willing to participate. Exclusion criteria were as follows: (1) patients who were treated 3 months before admission, (2) patients with multiple diseases, and (3) patients who failed to cooperate with researchers. The patient group included 32 males and 66 females, and age range from 30 to 64 years old, with a mean age of 48.2 ± 6.1 years old. Patients were staged according to following methods: stage 1: age at 30 to 35 years old without visible symptoms; stage 2: after age at 35 years old, bone breakdown happens faster than bone buildup, no visible symptoms, only can be detected through bone-density tests; stage 3: age at 45 to 55, bones become so and may break from normal stress; stage 4: bone fractures continue, pain increases, and may cause disability. There were 20 cases at stage I, 28 cases at stage II, 22 cases at stage III, and 28 cases at stage IV. The control group included 22 males and 38 females, and the age range from 30 to 65 years old, with a mean age of 48.7 ± 5.7 years old. No significant differences in age and gender were found between patient and control groups. This study passed the review of Baoding First Central Hospital, and all participants signed informed consent.

Bone marrow osteoclast precursors were isolated from C57Bl/6 J mice (8 weeks old, Guangdong Medical Experimental Animal Center, Guangdong, China). Primary marrow–derived osteoclasts were generated from bone marrow osteoclast precursors. All operations here were performed in strict accordance with the methods described by Stiffel et al. [11].

Total RNA was extracted from plasma using RNAzol® RT RNA Isolation Reagent (Sigma-Aldrich). High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) was used to performed reverse transcription. To detect the expression of lncRNA TUG1 and PTEN mRNA, Luna® Universal One-Step RT-qPCR Kit (NEB) was used to prepare PCR reaction systems. Primers of lncRNA TUG1 and β-actin were designed and synthesized by GenePharma (Shanghai, China). The expression of lncRNA TUG1 was normalized to endogenous controls β-actin using the 2^−ΔΔCT method.

Vectors expressing lncRNA TUG1 and empty vectors were designed and constructed by GenePharma (Shanghai, China). LncRNA TUG1 siRNA and scrambled negative control siRNA were also designed and constructed by GenePharma (Shanghai, China). Lipofectamine 2000 reagent (Thermo Fisher Scientific) was used to transfect vectors and siRNAs into primary marrow–derived osteoclasts with vectors at a dose of 10 nM and siRNAs at a dose of 50 nM. Cells only treated with lipofectamine 2000 reagent were control cells. Cells transfected with empty vectors or scrambled negative control siRNA were negative control cells.

Expression of lncRNA TUG1 was detected at 24 h after transfection, and cell proliferation was only detected in cases of over-expression rate of lncRNA TUG1 reached 200% and knockdown rate reached 50%. Briefly, primary marrow–derived osteoclasts were harvested and single-cell suspensions were prepared with a cell density of 3 × 10⁴ cells/ml. Cells were transferred to a 96-well plate with 0.1 ml in each well. Cells were cultivated under normal conditions (37 °C, 5% CO₂), followed by the addition of CCK-8 solution (10ul, Sigma-Aldrich) 24, 48, 72, and 96 h later. Cells were then cultivated for an additional 4 h, and OD values 450 nm were measured to calculate cell proliferation rate.

Expression of lncRNA TUG1 was detected at 24 h after transfection, and cell apoptosis was only detected in cases of over-expression rate of lncRNA TUG1 reached 200% and knockdown rate reached 50%. Briefly, primary marrow–derived osteoclasts were harvested and single-cell suspensions were prepared with a cell density of 3 × 10⁴ cells/ml using serum-free medium. Ten-milliliter cell suspension was added into each well of a 6-well plate, and 0.25% trypsin digestion was performed. After cells were cultivated for 48 h, staining with Annexin V-FITC (Dojindo, Japan) and propidium iodide (PI) was performed and cell apoptosis was detected by flow cytometry.

All experiments were repeated 3 times, and the mean ± standard deviation was calculated. The unpaired t test was used for comparisons between 2 groups, and one-way ANOVA followed by Tukey’s test was performed to compare 3 groups. Diagnostic values of lncRNA CASC11 for osteoclasts were performed by the receiver operating characteristic (ROC) curve with osteoporosis patients as true positive cases and healthy participants as true negative cases. Differences with p < 0.05 were statistically significant.

Expression of lncRNA TUG1 in plasma of 98 patients with osteoporosis and 60 healthy participants was detected by RT-qPCR. Compared with healthy participants, plasma levels of lncRNA TUG1 were significantly higher in osteoporosis patients (Fig. 1, p < 0.05).

Diagnostic values of lncRNA CASC11 for osteoclasts were performed by the ROC curve with osteoporosis patients as true positive cases and healthy participants as true negative cases. As shown in Fig. 2, the area under the curve was 0.90, with a standard error of 0.023 and a 95% confidence interval of 0.86–0.95. ROC curve analysis showed that the upregulation of plasma lncRNA TUG1 distinguished osteoporosis patients from healthy participants.

Among 98 patients with osteoporosis, there were 20 cases at stage I, 28 cases at stage II, 22 cases at stage III, and 28 cases at stage IV. As shown in Fig. 3, plasma levels of lncRNA TUG1 were significantly increased with an increase in stages (p < 0.05).

Over-expression and siRNA silencing experiments were performed to investigate the role of lncRNA TUG1 in the regulation of the proliferation and apoptosis of mice osteoclasts. Compared with control and negative control groups, over-expression of lncRNA TUG1 significantly promoted the proliferation (Fig. 4a, p < 0.05) and inhibited the apoptosis (Fig. 4b, p < 0.05) of mice osteoclasts. In addition, lncRNA TUG1 siRNA silencing played the opposite role. Moreover, lncRNA TUG1 over-expression led to downregulated PTEN mRNA, while lncRNA TUG1 siRNA silencing played an opposite role (Fig. 4c, p < 0.05).